The determination of the concentration of particles in a sample is often used, e.g. in connection with diagnosing a patient, where the concentration of white blood cells in a sample is one parameter used for determining the actual disease, or in connection with monitoring the state of a machine where the number of particles in a sample of oil from the engine may give an indication of any upcoming problems before they get critical.
Determination of the concentration of particles in a sample may be done by a number of methods. One of the methods is flow cytometry. Flow cytometry requires rather expensive equipment, firstly because the flow rate must be controlled and measured with very high accuracy to get a sufficiently precise measure of the volume, secondly because the detection system must work at short acquisition times in order to get reliable data from the particles present in the detector as they pass by. Laor (US 2006/0084125) describes a system for detection biological particles in a liquid sample where the liquid sample is flowing through a sample compartment and an object plane of an optical detection device has a non-zero angle to the flow direction.
Another method for determination of the concentration of particles in a sample is by microscopically viewing the sample either for manual or for automated detection and counting the particles confined in a certain well known volume. In patent application WO 2008/010761 by Olesen et al. such a method and apparatus is presented. In this method a portion of the sample is imaged onto an image recorder such as a 2-dimensional CCD-camera and the image is created by sending light through the sample towards the image recorder. The thickness of the imaged part of the sample is limited as the particles must be viewable and detectable through the sample. If the sample is too thick the light will be scattered and absorbed in the sample creating a poor quality image. Some of the particles in the sample may even be in the shadow of other particles making an accurate count difficult or impossible. The size of the image will be limited by the resolution of the image recorder and thereby the volume of the sample that may be used in the detection and counting of the particles will be limited. This is not a serious problem as long as the concentration of the particles to be counted is fitted to the size of the volume and the particle size. But if the concentration is high, an accurate measure may be difficult or impossible to determine. In this case a dilution of the sample could solve the measurement problem, but this knowledge may not be present until the measurement has been carried out. If the concentration is low the statistics for the measure will be poor, as small deviations in the count of particles or small deviations in the size of the volume may have great influence on the result. In this case the measurement should be carried out over a larger volume. Especially when using the method proposed by Olesen et al. in WO 2008/010761 for determining the distribution of different white blood cells the method may fall short. In this case it is important to have good statistics, but the sample volume is limited and if one or more of the white blood cell types have a low count, the statistical certainties may be poor.
In US 2008/0100703 Yamada describes a microscope system which makes a focus map of a sample with a large area compared to the area that can be imaged by the microscope. The information from the focus map is used when acquiring images of the different regions of the sample. These images are subsequently combined to provide a large scale image of the sample. The images of different sample regions are acquired by taking a plurality of images of one region at different depths and translating the sample and detection system relative to each other before images of another region is acquired. The translation of the sample and optical detection assembly relative to each other is parallel to the object plane of the optical detection assembly, i.e. the optical axis and the scanning path are perpendicular to each other, and the surface of the sample device is parallel to the object plane i.e. the normal of the surface is parallel to the optical axis.